Fluorescence quantum yields of matrices used in ultraviolet matrix-assisted laser desorption/ionization.

RATIONALE: Ultraviolet matrix-assisted laser desorption/ionization (MALDI) is among the most popular soft ionization methods in mass spectrometry. Several theoretical models have been proposed to explain the primary ion generation in MALDI. These models require knowledge of various matrix molecular parameters for simulation. One such parameter is the fluorescence quantum yield. However, the fluorescence quantum yield reported in previous studies remains controversial. METHODS: In this study, we used a commercial and a homemade integrating sphere to measure the absorption and fluorescence quantum yields of several commonly used matrices, including 2,3-dihydroxybenzoic acid, 2,4-dihydroxybenzoic acid (2,4-DHB), 2,5-dihydroxybenzoic acid (2,5-DHB), 2,6-dihydroxybenzoic acid, 3,4-dihydroxybenzoic acid, 3,5-dihydroxybenzoic acid, α-cyano-4-hydroxycinnamic acid, 2,4,6-trihydroxyacetophenone, and ferulic acid. RESULTS: The fluorescence quantum yields of these matrices were determined to be low (<0.08) at low laser fluences and decreased as the laser fluence increased. The fluorescence quantum yields at the typical laser fluence for MALDI are below 0.04 (2,4-DHB and 2,5-DHB) and 0.01 (the other matrices). Shot-to-shot fluctuations of fluorescence intensity and absorption are not directly related to the fluctuation of ions. Possible mechanisms for the decrease in the fluorescence quantum yield as the laser fluence increased were discussed. CONCLUSIONS: The fluorescence quantum yields of these commonly used matrices are much lower than those reported in previous studies. Although fluorescence quantum yield is an important parameter and it is crucial to obtain an accurate value for theoretical models in simulations, the use of fluorescence quantum yield alone is not a sufficient parameter to justify these models.

Unexpected Dissociation Mechanism of Sodiated N-Acetylglucosamine and N-Acetylgalactosamine.

The mechanism for the collision-induced dissociation (CID) of two sodiated N-acetylhexosamines (HexNAc), N-acetylglucosamine (GlcNAc), and N-acetylgalactosamine (GalNAc), was studied using quantum-chemistry calculations and resonance excitation in a low-pressure linear ion trap. Experimental results show that the major dissociation channel of the isotope labeled [1-18O, D5]-HexNAc is the dehydration by eliminating HDO, where OD comes from the OD group at C3. Dissociation channels of minor importance include the 0,2A cross-ring dissociation. No difference has been observed between the CID spectra of the α- and ß-anomers of the same HexNAc. At variance, the CID spectra of GlcNAc and GalNAc showed some differences, which can be used to distinguish the two structures. It was observed in CID experiments involving disaccharides with a HexNAc at the nonreducing end that a ß-HexNAc shows a larger dissociation branching ratio for the glycosidic bond cleavage than the α-anomer. This finding can be exploited for the rapid identification of the anomeric configuration at the glycosidic bond of HexNAc-R' (R' = sugar) structures. The experimental observations indicating that the dissociation mechanisms of HexNAcs are significantly different from those of hexoses were explained by quantum-chemistry calculations. Calculations show that ring opening is the major channel for HexNAcs in a ring form. After ring opening, dehydration shows the lowest barrier. In contrast, the glycosidic bond cleavage becomes the major channel for HexNAcs at the nonreducing end of a disaccharide. This reaction has a lower barrier for ß-HexNAcs as compared with the barrier of the corresponding α-anomers, consistent with the higher branching ratio for ß-HexNAcs observed in experiment.

Toward Closing the Gap between Hexoses and N-Acetlyhexosamines: Experimental and Computational Studies on the Collision-Induced Dissociation of Hexosamines.

Motivated by the fundamental difference in the reactivity of hexoses and N-acetylhexosamines under collision-induced dissociation (CID) mass spectrometry conditions, we have investigated the CID of two hexosamines, glucosamine (GlcN) and galactosamine (GalN), experimentally and computationally. Both hexosamines undergo ring-opening and then dissociate via the 0,2A and the 0,3A (0,3X) cross-ring cleavage channels. The preference for the ring-opening is similar to the behavior of N-acetylhexosamines and explains why the two anomers of the same sugar give the same mass spectrum. While the spectrum for GlcN is dominated by the 0,2A signal, the signal intensities for both 0,2A and the 0,3A (0,3X) dissociation channels are comparable for GalN, which allows GlcN and GalN to be distinguished easily. Calculations at MP2 level of theory indicate that this is related to the differences in the relative barrier heights for the 0,2A and the 0,3A (0,3X) cross-ring cleavage channels. This, in return, reflects the circumstance that the 0,2A cross-ring cleavage barriers are different for the two sugars, while the barriers of all other dissociation channels are comparable. While the mechanisms of the cross-ring dissociation channels of hexoses are well described using the retro-aldol mechanism in the literature, this study proposes a new mechanism for the 0,3A (0,3X) cross-ring cleavage of hexosamines that involves the formation of an epoxy intermediate or a zwitterionic intermediate.

Is energy pooling necessary in ultraviolet matrix-assisted laser desorption/ionization?

RATIONALE: Energy pooling has been suggested as the key process for generating the primary ions during ultraviolet matrix-assisted laser desorption/ionization (UV-MALDI). In previous studies, decreases in fluorescence quantum yields as laser fluence increased for 2-aminobenzoic acid, 2,5-dihydroxybenzoic acid (2,5-DHB), and 3-hydroxypicolinic acid were used as evidence of energy pooling. This work extends the research to other matrices and addresses whether energy pooling is a universal property in UV-MALDI. METHODS: Energy pooling was investigated in a time-resolved fluorescence experiment by using a short laser pulse (355 nm, 20 ps pulse width) for excitation and a streak camera (1 ps time resolution) for fluorescence detection. RESULTS: The excited-state lifetime of 2,5-DHB decreased with increases in laser fluence. This suggests that a reaction occurs between two excited molecules, and that energy pooling may be one of the possible reactions. However, the excited-state lifetime of 2,4,6-trihydroxyacetophenone (THAP) did not change with increases in laser fluence. The upper limit of the energy pooling rate constant for THAP is estimated to be approximately 100-500 times smaller than that of 2,5-DHB. CONCLUSIONS: The small energy pooling rate constant for THAP indicates that the potential contribution of the energy pooling mechanism to the generation of THAP matrix primary ions should be reconsidered.

Fluorescence spectroscopy of UV-MALDI matrices and implications of ionization mechanisms.

Matrix-assisted laser desorption ionization (MALDI) has been widely used in the mass analysis of biomolecules; however, there are a lot of debates about the ionization mechanisms. Previous studies have indicated that S1-S1 annihilation might be a key process in the generation of primary ions. This study investigates S1-S1 annihilation by examining the time-resolved fluorescence spectra of 12 matrices. No S1-S1 annihilation was observed in six of these matrices (3-hydroxy-picolinic acid, 6-aza-2-thiothymine, 2,4-dihydroxy-acetophenone, 2,6-dihydroxy-acetophenone, 2,4,6-trihydroxy-acetophenone, and ferulic acid). We observed two matrix molecules reacting in an electronically excited state (S1) in five of these matrices (2,5-dihydroxybenzoic acid, α-cyano-4-hydroxycinnamic acid, 2,5-dihydroxy-acetophenone, 2,3-dihydroxybenzoic acid, and 2,6-dihydroxybenzoic acid), and S1-S1 annihilation was a possible reaction. Among these five matrices, no S1-S1 annihilation was observed for 2,3-dihydroxybenzoic acid in typical peak power region of nanosecond laser pulses in MALDI, but a very small value of reaction rate constant was observed only in the high peak power region. The excited-state lifetime of sinapinic acid was too short to determine whether the molecules reacted in an electronically excited state. No correlation was observed between the ion generation efficiency of MALDI and S1-S1 annihilation. The results indicate that the proposal of S1-S1 annihilation is unnecessary in MALDI and energy pooling model for MALDI ionization mechanism has to be modified.

Structural Determination of Polysaccharides Lichenin Using Logically Derived Sequence Tandem Mass Spectrometry.

A new mass spectrometry method, logically derived sequence (LODES) tandem mass spectrometry (MSn), was applied to determine the primary structure of polysaccharide lichenin. Conventional polysaccharide structural analysis requires complex processes, including derivation, permethylation, gas chromatography-mass spectrometry, and nuclear magnetic resonance spectrometry. Many of these processes can be replaced by LODES/MSn. In this new method, polysaccharides are hydrolyzed into monosaccharides, disaccharides, and oligosaccharides, and structures of these molecules are determined using LODES/MSn. The application of LODES/MSn for determination of primary structure of polysaccharide lichenin was demonstrated. The repeating unit of lichenin was determined to be An-Bn, where A represents ß-Glc-(1 → 4)-ß-Glc-(1 → 4)-ß-Glc-(1 → 3)-Glc, B represents ß-Glc-(1 → 4)-ß-Glc-(1 → 4)-ß-Glc-(1 → 4)-ß-Glc-(1 → 3)-Glc, n is an integral, and n ≥ 2 exists but n = 1 cannot be excluded. LODES/MSn, which substantially reduces the time, effort, and sample quantity necessary for structural determination of oligosaccharides, is a powerful tool for polysaccharide primary structural determination.

Temperature Dependence of Desorbed Ions and Neutrals and Ionization Mechanism of Matrix-Assisted Laser Desorption/Ionization.

Two separate temperature-dependent experiments were performed to investigate the ionization mechanism of ultraviolet matrix-assisted laser desorption/ionization (UV-MALDI) of matrix 2,5-dihydroxybenzoic acid (2,5-DHB). First, the angular resolved intensity and velocity distributions of neutrals desorbed from the 2,5-DHB solid sample through UV laser (355 nm) pulse irradiation were measured using a rotating quadrupole mass spectrometer. Second, the desorbed neutrals, at an angle normal to the surface, and the desorbed ions were simultaneously detected for each laser shot using the quadrupole mass spectrometer and a time-of-flight mass spectrometer, respectively. Both experiments were conducted at two initial temperatures: 100 and 300 K. The measurements from these two experiments were used to calculate the initial temperature dependence of the ion-to-neutral ratio. The results closely agreed with the predictions of the temperature-dependent ion-to-neutral ratio using the thermal model, indicating that thermally induced proton transfer is the dominant reaction that generates initial ions of 2,5-DHB in UV-MALDI.

Formation of Metal-Related Ions in Matrix-Assisted Laser Desorption Ionization.

In a study of the metal-related ion generation mechanism in matrix-assisted laser desorption ionization (MALDI), crystals of matrix used in MALDI were grown from matrix- and salt-containing solutions. The intensities of metal ion and metal adducts of the matrix ion obtained from unwashed crystals were higher than those from crystals washed with deionized water, indicating that metal ions and metal adducts of the matrix ions are mainly generated from the surface of crystals. The contributions of preformed metal ions and metal adducts of the matrix ions inside the matrix crystals were minor. Metal adducts of the matrix and analyte ion intensities generated from a mixture of dried matrix, salt, and analyte powders were similar to or higher than those generated from the powder of dried droplet crystals, indicating that the contributions of the preformed metal adducts of the matrix and analyte ions were insignificant. Correlation between metal-related ion intensity fluctuation and protonated ion intensity fluctuation was observed, indicating that the generation mechanism of the metal-related ions is similar to that of the protonated ions. Because the thermally induced proton transfer model effectively describes the generation of the protonated ions, we suggest that metal-related ions are mainly generated from the salt dissolution in the matrix melted by the laser. Graphical Abstract ᅟ.

Ion intensity and thermal proton transfer in ultraviolet matrix-assisted laser desorption/ionization.

The ionization mechanism of ultraviolet matrix-assisted laser desorption/ionization (UV-MALDI) was investigated by measuring the total cation intensity (not including sodiated and potasiated ions) as a function of analyte concentration (arginine, histidine, and glycine) in a matrix of 2,4,6-trihydroxyacetophenone (THAP). The total ion intensity increased up to 55 times near the laser fluence threshold as the arginine concentration increased from 0% to 1%. The increases were small for histidine, and a minimal increase occurred for glycine. Time-resolved fluorescence intensity was employed to investigate how analytes affected the energy pooling of the matrix. No detectable energy pooling was observed for pure THAP and THAP/analyte mixtures. The results can be described by using a thermal proton transfer model, which suggested that thermally induced proton transfer is crucial in the primary ion generation in UV-MALDI.